The histone code is comprised of many post-translational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. Small molecule probes that disrupt the interaction between histone binding proteins and chromatin would enable a systematic chemical biology study of proteins that read the histone code and could potentially reveal new targets for drug discovery. However, no such small molecules are known. This dearth of quality molecular probes can largely be attributed to the novelty of the target class and the lack of screening assays. We have developed the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins and propose that it be used to initiate a full-scale discovery effort for antagonists of histone binding proteins. In this work, we focused on the development of an AlphaScreen"-based assay for Malignant Brain Tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail peptides. This assay takes advantage of the avidity of the AlphaScreen" beads to clear the hurdle to assay development presented by the low micromolar binding constants of the histone binding proteins for their cognate peptides. The assay was demonstrated on L3MBTL1, a prototypical MBT-domain containing protein, and was capable of detecting its binding to a 15-mer peptide representative of monomethylated lysine 9 on histone H3, H3K9Me1. In the assay, binding partners were present in the nanomolar range, well below their actual Kd, but accurate measurement of IC50's of competitor peptides was possible under these conditions. The assay was stable over 24 h, and had a Z'value of 0.87 in a 384- well low volume format and can be easily adapted to 1536 well plates. The assay is applicable to other families of methyl-lysine binding proteins and has been used to create a panel of 5 assays that can be used for probe characterization and optimization. PUBLIC HEALTH RELEVANCE: This project will create small molecule probes that will be used to interrogate the function of methyl lysine binding domains (specifically Malignant Brain Tumor (MBT) domains) in chromatin function. These probes will allow researchers to establish connections between disease states and MBT containing proteins.